Recent studies have begun to elucidate the antigenic relationships among human rotaviruses on the basis of the antigenic specificity of the VP4 protein. A VP4 serotype system was recently established by the neutralization technique using guinea pig antiserum to the baculovirus recombinant-expressed VP4 protein of human rotavirus (HRV) strain KU, DS-1, 1076, or K8. The criterion for serotype specificity was a greater than eight-fold reciprocal difference in neutralizing antibody titer. Three distinct VP4 serotypes were defined, namely VP4 1A and 1B. In a recent study, the VP5 and VP8 cleavage subunits of VP4 from three different human rotavirus strains, KU (VP4 serotype 1A), DS-1 (VP4 serotype 1B), and 1076 (VP4 serotype 2), were independently expressed in Escherichia coli. Guinea pig antisera produced in response to immunization with these recombinant VP5 and VP8 proteins were used in cross-neutralization tests to determine the contribution of each of these two subunits to the overall antigenic specificity of VP4. In addition, three peptides which collectively span the entire VP8 subunit of VP4 were also expressed in E. coli, and each peptide was analyzed by the immunoblot technique using different VP4 serotype antisera. Guinea pig antisera produced in response to immunization with these peptides were used in plaque reduction neutralization (PRN) assay to assess their specificity. The distribution of conserved as well as specific epitopes in the VP8 subunit of VP4 was studied.